Two New Sesquiterpenoids and a New Shikimic Acid Metabolite from Mangrove Sediment-Derived Fungus Roussoella sp. SCSIO 41427

Two new sesquiterpenoid derivatives, elgonenes M (1) and N (2), and a new shikimic acid metabolite, methyl 5-O-acetyl-5-epi-shikimate (3), were isolated from the mangrove sediment-derived fungus Roussoella sp. SCSIO 41427 together with fourteen known compounds (4–17). The planar structures were elucidated through nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analyses. The relative configurations of 1–3 were ascertained by NOESY experiments, while their absolute configurations were determined by electronic circular dichroism (ECD) calculation. Elgonene M (1) exhibited inhibition of interleukin-1β (IL-1β) mRNA, a pro-inflammatory cytokine, at a concentration of 5 μM, with an inhibitory ratio of 31.14%. On the other hand, elgonene N (2) demonstrated inhibition at a concentration of 20 μM, with inhibitory ratios of 27.57%.


Introduction
As a defensive response to injury, inflammation is not always harmful; however, excessive expression of inflammatory mediators can lead to immune system dysregulation, resulting in inflammatory diseases [1].Currently, the most widely used anti-inflammatory drugs worldwide are nonsteroidal anti-inflammatory drugs (NSAIDs), but they come with serious gastrointestinal side effects and cardiovascular risks [2].Therefore, exploring new anti-inflammatory drugs is crucial research.Scientists are actively investigating natural sources, including marine organisms, plants, and microorganisms, in the quest for new therapeutic agents with potent anti-inflammatory properties and improved safety profiles.
The ocean is a treasure trove of resources, and the search for new natural products from the marine environment for drug development has become an international research hotspot.Statistics show that, in the year 2021 alone, researchers discovered 1425 new compounds with a wide range of biological activities from marine-derived organisms [3].Among these, mangrove sediment-derived microorganisms (MSMs) are an important source of various natural products.As of 2021, researchers have isolated and identified 519 new natural products from MSMs, with 57% of these compounds originating from fungi and exhibiting broad and effective biological activities.These compounds from marine-derived fungi have shown promising antimicrobial properties against various between the side chain structure and the cyclohexene ring was revealed through HMBC correlations from H-2/H2-6 to C-7, H-5 to C-9/C-10.Furthermore, 1 H-1 H COSY correlations between H-2/H-3/H-4/H-5/H-6 confirmed the cyclohexyl structure.The above NMR data indicated that the structural skeleton of 3 was identical to that of a synthesized compound, methyl 5-O-acetyl-5-epi-shikimate [11].The NOESY correlations of H-3/H-5, H-3/H-4, and H-4/H-5 supposed that the relative configuration of 3 was rel-(3R, 4R, 5S).Finally, its absolute configuration was determined to be 3R, 4R, 5S through the ECD calculations and specific rotation compared with 15 (Figure 3).

Structural Determination
Compound 1 was obtained as a pale yellow oil, and its molecular formula of C 15 H 22 O 3 was deduced from the negative HRESIMS ion peak at m/z 249.1499 [M-H] − (calculated for C 15 H 21 O 3 − , 249.1496), implying five degrees of hydrogen deficiency.The 1 H NMR data (Table 1) showed three methyls at δ H 1.33 (s, H 3 -14), 1.64 (s, H 3 -13), and 1.96 (s, H 3 -15); three methylenes at δ H 1.81/2.03(H 2 -8), 1.99 (m, H 2 -11), and 1.24/1.90(H 2 -12); and five olefinic protons at 7.29 (d, J = 11.4Hz, H-3), 6.59 (dd, J = 11.4,15.1 Hz, H-4), 6.17 (d, J = 15.1 Hz, H-5), 1.63 (m, H-7), and 5.36 (m, H-9).Analysis of the 13  ).The 1 H-1 H COSY correlations (Figure 2) of H 2 -11/H 2 -12/H 2 -7/H 2 -8/H-9 and the HMBC correlations from H 3 -13 to C-9, C-10, and C-11 revealed a six-membered ring with a methyl at C-10.A chain system with hydroxyl and carboxyl groups was confirmed by the 1 H-1 H COSY correlations of H-3/H-4/H-5 and the HMBC correlations from H-2 to C-1 and C-4, as well as from H-4 to H-6 and from H-5 to H 3 -14.The HMBC correlation from H 3 -14 to C-7 suggested that methine carbon C-6 at the end of the chain was located at C-7 on the ring.Furthermore, the NMR data indicated that the planar structure of 1 was similar to that of the known compound, sesquiterpene elgonene D [12].Also, Compound 2 was obtained as a pale yellow oil, and its molecular formula of C 15 H 22 O 3 was deduced from the negative HRESIMS ion peak at m/z 249.1498 [M-H] − (calculated for C 15 H 21 O 3 − , 249.1496).The NMR spectroscopic data (Table 1) comparison between 2 and 1 revealed that they possess identical planar structures.As for their configuration, ∆ 2,3 and ∆ 4,5 double bonds in 1 and 2 were both deduced as E by the NOESY correlation of H 3 -15/H-3 (Figure 2) and the large coupling constant J H-4/H-5 = 15.1/15.2Hz.Due to the consistent trends observed in their experimental CD curves, the ECD calculations of 1/2 (Figure 3) indicated that the configuration was established as 6R,7S/6R,7R.Thus, compounds 1 and 2 are a pair of diastereomers, with both having an R configuration of C-6.To differentiate between the diastereomers 1 and 2, a detailed conformational analysis was performed.The dominant conformation of the 6R,7S stereoisomer was characterized by a close spatial proximity between H 3 -14 and H-7, indicating the presence of a NOESY effect of H 3 -14/H-7 (Figure 2).Compared with the NMR experimental data, the NOESY correlation of H 3 -14/H-7 in 1 suggested its absolute configuration as 6R,7S.Similarly, the absence of a NOESY correlation of H 3 -14/H-7 in 2 suggested its absolute configuration as 6R,7R.Therefore, the gross structures, as depicted in Figure 1, were constructed and have been designated as elgonenes M (1) and N (2).

Bioactive Assay
Most acute and chronic non-infectious inflammatory diseases are associated with the pro-inflammatory cytokine interleukin-1β (IL-1β), and clinical studies have proven that blocking IL-1β can effectively resolve inflammation [25].In order to investigate the antiinflammatory effects of the compounds, an inflammation model was established using lipopolysaccharide (LPS)-stimulated C57BL/6 mouse primary bone marrow-derived macrophages.Dexamethasone, a well-known anti-inflammatory drug, was used as a positive control for comparison.The experimental results revealed that both 1 (5 μM) and 2 (10 and 20 μM) exerted noticeable effects on the expression of endogenous inflammatory factor IL-1β mRNA within the cells.Specifically, Compound 1 at 5 μM significantly reduced the expression of IL-1β mRNA, resulting in an impressive inhibition rate of 31.14%.Similarly, Compound 2 at a concentration of 20 μM exhibited a significant reduction in the

Bioactive Assay
Most acute and chronic non-infectious inflammatory diseases are associated with the pro-inflammatory cytokine interleukin-1β (IL-1β), and clinical studies have proven that blocking IL-1β can effectively resolve inflammation [25].In order to investigate the antiinflammatory effects of the compounds, an inflammation model was established using lipopolysaccharide (LPS)-stimulated C57BL/6 mouse primary bone marrow-derived macrophages.Dexamethasone, a well-known anti-inflammatory drug, was used as a positive control for comparison.The experimental results revealed that both 1 (5 μM) and 2 (10 and 20 μM) exerted noticeable effects on the expression of endogenous inflammatory factor IL-1β mRNA within the cells.Specifically, Compound 1 at 5 μM significantly reduced the expression of IL-1β mRNA, resulting in an impressive inhibition rate of 31.14%.Similarly, Compound 2 at a concentration of 20 μM exhibited a significant reduction in the level of IL-1β mRNA, with an inhibition rate of 27.57% (Figure 4).These findings sug- The connection between the side chain structure and the cyclohexene ring was revealed through HMBC correlations from H-2/H 2 -6 to C-7, H-5 to C-9/C-10.Furthermore, 1 H-1 H COSY correlations between H-2/H-3/H-4/H-5/H-6 confirmed the cyclohexyl structure.The above NMR data indicated that the structural skeleton of 3 was identical to that of a synthesized compound, methyl 5-O-acetyl-5-epi-shikimate [11].The NOESY correlations of H-3/H-5, H-3/H-4, and H-4/H-5 supposed that the relative configuration of 3 was rel-(3R, 4R, 5S).Finally, its absolute configuration was determined to be 3R, 4R, 5S through the ECD calculations and specific rotation compared with 15 (Figure 3).

Bioactive Assay
Most acute and chronic non-infectious inflammatory diseases are associated with the pro-inflammatory cytokine interleukin-1β (IL-1β), and clinical studies have proven that blocking IL-1β can effectively resolve inflammation [25].In order to investigate the anti-inflammatory effects of the compounds, an inflammation model was established using lipopolysaccharide (LPS)-stimulated C57BL/6 mouse primary bone marrow-derived macrophages.Dexamethasone, a well-known anti-inflammatory drug, was used as a positive control for comparison.The experimental results revealed that both 1 (5 µM) and 2 (10 and 20 µM) exerted noticeable effects on the expression of endogenous inflammatory factor IL-1β mRNA within the cells.Specifically, Compound 1 at 5 µM significantly reduced the expression of IL-1β mRNA, resulting in an impressive inhibition rate of 31.14%.Similarly, Compound 2 at a concentration of 20 µM exhibited a significant reduction in the level of IL-1β mRNA, with an inhibition rate of 27.57% (Figure 4).These findings suggested that both 1 and 2 possessed anti-inflammatory properties.Compound 1 showed activity only at 5 µM and was inactive at higher concentrations.The cellular toxicity of 1 on LPS-stimulated C57BL/6 mouse primary bone marrow-derived macrophages at 10 and 20 µM was speculated to be the reason behind these results.Therefore, the difference in activity between compounds 1 and 2 may be attributed to their distinct configurations at C-7.In addition, anti-inflammatory activities have also been found in sesquiterpene derivatives in the published literature [26,27], proving that these types of compounds indeed have certain research value in anti-inflammatory activity.Further studies are needed to elucidate the underlying mechanisms and explore their potential in the treatment of inflammatory diseases.
During the in vitro antitumor activity screening, compounds 1, 3-6, 8-10, and 14 were evaluated at a concentration of 50 µM.The results indicated that the inhibition rate on MDA-MB-435 tumor cells (human breast cancer cells) was below 50%, indicating that their IC 50 values were greater than 50 µM.activity between compounds 1 and 2 may be attributed to their distinct configurations at C-7.In addition, anti-inflammatory activities have also been found in sesquiterpene derivatives in the published literature [26,27], proving that these types of compounds indeed have certain research value in anti-inflammatory activity.Further studies are needed to elucidate the underlying mechanisms and explore their potential in the treatment of inflammatory diseases.During the in vitro antitumor activity screening, compounds 1, 3-6, 8-10, and 14 were evaluated at a concentration of 50 μM.The results indicated that the inhibition rate on MDA-MB-435 tumor cells (human breast cancer cells) was below 50%, indicating that their IC50 values were greater than 50 μM.

General Experimental Procedures
The UV and ECD spectra was recorded on a UV-Vis spectrophotometer model 8453VU-Vis (Agilent, Beijing, China) and a chirascan circular dichroism spectrometer (Applied Photophysics, Surrey, Britain), respectively.The IR spectrum was obtained using an IR Affinity-1 spectrometer (Shimadzu, Beijing, China).HRESIMS spectra were recorded with a Bruker maXis Q-TOF mass spectrometer.The NMR spectra were recorded on a AVANCE III HD 600 MHz spectrometer (Bruker BioSpin International AG, Fällanden, Switzerland), and chemical shifts were recorded as δ-values.High-Performance Liquid Chromatograph (HPLC) was performed on the Agilent 1260 with a DAD detector using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm).Thin-layer chromatography analysis (TLC) and column chromatography (CC) were carried out on plates precoated with silica gel GF254 (10-40 μm), over silica gel (200-300 mesh) (Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden).Spots were detected on TLC (Qingdao Marine Chemical Factory) under 254 nm UV light.All solvents used, except for the liquid chromatography mobile phase, were of analytical grade (Tianjin Fuyu Chemical and Industry Factory, Tianjin, China).The mobile phase for liquid chromatography was of HPLC gradient grade (Shanghai Xingke High Purity Solvents Co., Ltd, Shanghai, China.).

Fungal Source and Strain Identification
The fungal strain SCSIO 41427 was isolated from a mangrove sediment sample collected from a Gaoqiao mangrove in Lianjiang, China.The strains were stored on MB agar (malt extract 15 g, sea salt 10 g, agar 16 g, H2O 1 L, pH 7.4-7.8)slants in liquefied petrola-

General Experimental Procedures
The UV and ECD spectra was recorded on a UV-Vis spectrophotometer model 8453VU-Vis (Agilent, Beijing, China) and a chirascan circular dichroism spectrometer (Applied Photophysics, Surrey, Britain), respectively.The IR spectrum was obtained using an IR Affinity-1 spectrometer (Shimadzu, Beijing, China).HRESIMS spectra were recorded with a Bruker maXis Q-TOF mass spectrometer.The NMR spectra were recorded on a AVANCE III HD 600 MHz spectrometer (Bruker BioSpin International AG, Fällanden, Switzerland), and chemical shifts were recorded as δ-values.High-Performance Liquid Chromatograph (HPLC) was performed on the Agilent 1260 with a DAD detector using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 µm).Thin-layer chromatography analysis (TLC) and column chromatography (CC) were carried out on plates precoated with silica gel GF254 (10-40 µm), over silica gel (200-300 mesh) (Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden).Spots were detected on TLC (Qingdao Marine Chemical Factory) under 254 nm UV light.All solvents used, except for the liquid chromatography mobile phase, were of analytical grade (Tianjin Fuyu Chemical and Industry Factory, Tianjin, China).The mobile phase for liquid chromatography was of HPLC gradient grade (Shanghai Xingke High Purity Solvents Co., Ltd, Shanghai, China).

Fungal Source and Strain Identification
The fungal strain SCSIO 41427 was isolated from a mangrove sediment sample collected from a Gaoqiao mangrove in Lianjiang, China.The strains were stored on MB agar (malt extract 15 g, sea salt 10 g, agar 16 g, H 2 O 1 L, pH 7.4-7.8)slants in liquefied petrolatum and deposited at Key Laboratory of Tropical Marine Bio-resources and Ecology, Chinese Academy of Sciences.The strain SCSIO 41427 was designated as Roussoella sp., due to its ITS sequence (GenBank accession No. OR574981) homology with Roussoella sp.LT796863.1.

Fungal Cultivation and Fermentation
The fermentation of Roussoella sp.SCSIO 41427 was carried out using a solid-state culture medium.The preparation of the medium involved combining 180 mL of distilled water, 3 g of sea salt, and 150 g of rice in a 1000 mL conical flask.The MB seed solution was prepared by mixing 400 mL of distilled water, 8 g of sea salt, and 6 g of malt extract in a 1000 mL conical flask, with pH adjustment set to 7.4-7.8.Both media were sterilized by autoclaving at 121 • C for 30 min and allowed to cool.The strain was activated by inoculating it into MB agar medium (or PDA medium) after being stored in paraffin oil.The activation process occurred at 26 • C for 5 days (typically 5 days), after which agar sections containing the newly cultivated Roussoella sp.SCSIO 41427 were transferred to MB seed liquid.This mixture was then cultured in two bottles at 27 • C with agitation at 180 rpm for 48 h to obtain the seed liquid.The seed liquid was subsequently transferred to the solid rice culture medium and allowed to ferment at 26 • C for 30 days.This process was scaled up to 60 bottles, yielding 45 bottles of fermented material (6.75 kg in dry) from Roussoella sp.SCSIO 41427.

ECD Calculation of 1-3
Conformational analyses of 1-3 were carried out by Spartan'14 software (v1.1.4,Wavefunction, Irvine, CA, USA) using a Molecular Merck force field.The conformers with a Boltzmann population exceeding 1% were subsequently optimized by utilizing Gaussion09 (D.01, Pittsburgh, PA, USA) at the B3LYP/6-31G (d) level in methanol using the PCM model [28].The optimized stable conformers were chosen for further ECD calculations at the B3LYP/6-311G (d, p) level in methanol.The rotatory strengths for a total of 20 excited states were calculated.The overall ECD data were weighted by Boltzmann distribution, and the ECD curves and enantiomeric ECD curves were generated by GuassianView 6.0 software with a half-bandwidth of 0.33 eV, based on the Boltzmann-calculated contribution of each conformer after UV correction.

Anti-Inflammatory Assay
Femurs and tibias were harvested from 6-8 week old C57BL/6 mice, rinsed thrice with 2% antibiotic-containing PBS, and both ends of the bones were removed.Bone marrow cavities were flushed with PBS, and the cell suspension was passed through a 70 µm cell strainer into a centrifuge tube.The suspension was centrifuged at 500× g for 5 min at 4 • C, the supernatant was discarded, and red blood cells were lysed by adding red blood cell lysis buffer and incubating for 10 min.Following another centrifugation at 500× g for 5 min at 4 • C, the supernatant was discarded, and cells were resuspended in α-MEM containing 10% FBS.The cells were seeded in culture dishes and incubated in a 37 • C, 5% CO 2 incubator for 2 h.The medium was then collected in centrifuge tubes, centrifuged at 500× g for 5 min at 4 • C, and cells were resuspended in α-MEM containing 50 ng/mL M-CSF.Cells were seeded at a density of 2.5 × 10 6 cells/mL in a 24-well plate and cultured for 5 days for subsequent experiments.The cells were divided into a blank group, a model group, treatment groups with compounds 1 and 2 (at concentrations of 1 µM, 5 µM, 10 µM, and 20 µM), and a positive control group with dexamethasone (20 µM).Except for the blank group, all other groups were co-incubated with 100 ng/mL lipopolysaccharide (LPS) for 6 h.After 6 h, the IL-1β mRNA expression was measured.The culture medium was discarded, and the cells were washed twice with PBS.RNA was extracted using the Trizol method, and its concentration was determined with NanoDrop.RNA was reverse transcribed into cDNA using the EnzyArtisan reverse transcription kit.A 10 µL qPCR reaction was prepared according to the SYBR qPCR kit (EnzyArtisan) instructions and run on a ROCHE qPCR LightCycler96 instrument following the recommended protocol.The housekeeping gene GAPDH was used as an internal control to normalize the CT values, using the 2 −∆∆Ct formula.The results were shown as mean ± SD from three independent experiments.

Cytotoxicity Bioassay
The cytotoxicity of 1, 3-6, 8-10, and 14 against MDA-MB-435 (human breast cancer cells) was determined by assessing cell viability through the 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assay.Briefly, cells were seeded at a density of 5 × 10 3 cells per well in a 96-well plate and left to incubate overnight, followed by treatment with the compounds for the required duration.The optical density at 570 nm (OD 570 ) was measured using a Hybrid Multi-Mode Reader (Synergy H1, BioTek, Santa Clara, CA, USA).The experiment was independently repeated three times.